DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging

描述（由申请人提供）：DNA纳米标签：用于细胞内成像的明亮荧光标记和传感器了解人类疾病的分子基础对于开发具有最小副作用的有效疗法至关重要。健康和疾病状态下的基本生物学过程涉及细胞内和细胞表面的瞬时分子间（例如蛋白质-蛋白质或蛋白质-核酸）相互作用。直接成像这些相互作用在真实的时间提供了无与伦比的洞察力的亲和力和动力学的分子协会。绝大多数这样的实验是使用荧光显微镜和目的蛋白质与绿色荧光蛋白（GFP）或其他FP之间的融合构建体进行的。虽然这些融合蛋白已经取得了实质性的进展，但GFP的相对低的亮度和光稳定性阻碍了需要短成像时间或高灵敏度的应用，这是由于感兴趣的蛋白质的丰度低。该提案的主要目的是创建一类新的明亮的荧光标记物，其相对于GFP将表现出大大改善的亮度和光稳定性。这将通过合成由具有数十个共价连接的嵌入染料的支链DNA纳米结构组成的多发色团组装体来实现。这些DNA纳米标签的设计利用了50年来关于使用荧光嵌入染料检测DNA的知识，以及最近在DNA纳米结构的设计和合成方面的工作。纳米标签的高亮度源自它们非常大的有效消光系数，这是由于存在许多与每个DNA支架结合的染料。附加的较长波长染料的DNA末端的附件将导致有效的能量转移和整个光谱的可见光和近红外区域的荧光颜色的调谐。通过合理设计染料和DNA结构，纳米标签将在其生物化学和光化学稳定性方面进行优化。最后，将合成纳米标签-抗体缀合物，并测试其在酵母细胞表面和果蝇胚胎内的标记。总的来说，该提案结合了有机化学、单分子光谱学和荧光显微镜，以创建一类新的普遍有用的明亮荧光标记。项目叙述DNA纳米标签：用于细胞内成像的明亮荧光标记和传感器拟议的研究将导致一类新的荧光标记，其将以几乎任何颜色可用，并且可以连接到各种识别模块，以允许以非常低的浓度对细胞表面和细胞内目标进行染色。检测和跟踪细胞中单个分子的能力将大大提高我们对基本生物学过程以及健康和疾病状态之间分子水平差异的理解，最终允许开发更有效的治疗方法，副作用更少。

DESCRIPTION (provided by applicant): DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging Understanding the molecular basis for human disease is essential for developing effective therapeutics with minimal side effects. The fundamental biological processes underlying both healthy and diseased states involve transient intermolecular (e.g. protein-protein or protein-nucleic acid) interactions within and at the surface of cells. Direct imaging of these interactions in real time provides unparallelled insight into the affinity and kinetics of molecular association. The overwhelming majority of such experiments are done using fluorescence microscopy and fusion constructs between proteins of interest and green fluorescent protein (GFP) or other FPs. While substantial progress has been made with these fusion proteins, the relatively low brightness and photostability of GFP hinder applications that require either short imaging times or high sensitivity due to low abundance of the protein of interest. The main objective of this proposal is to create a new class of bright fluorescent labels that will exhibit greatly improved brightness and photostability relative to GFP. This will be accomplished by synthesizing polychromophore assemblies consisting of a branched DNA nanostructure with dozens of covalently attached intercalating dyes. The design of these DNA nanotags takes advantage of 50 years of knowledge concerning the use of fluorescent intercalating dyes for the detection of DNA as well as more recent work in the design and synthesis of DNA nanostructures. The high brightness of the nanotags derives from their very large effective extinction coefficients due to the presence of many dyes bound to each DNA scaffold. Attachment of additional longer wavelength dyes to the DNA termini will lead to efficient energy transfer and tuning of the fluorescence color throughout the visible and near-IR regions of the spectrum. The nanotags will be optimized in terms of their biochemical and photochemical stability through rational design of the dye and DNA structures. Finally, nanotag-antibody conjugates will be synthesized and tested for labeling of yeast cell surfaces and within Drosophila embryos. Overall, this proposal combines organic chemistry, single- molecule spectroscopy and fluorescence microscopy to create a new class of generally useful, bright fluorescent labels. Project Narrative DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging The proposed research will lead to a new class of fluorescent labels that will be available in virtually any color and can be attached to various recognition modules to allow staining of cell surface and intracellular targets present at very low concentration. The ability to detect and track single molecules in cells will significantly advance our understanding of fundamental biological processes and the molecular-level distinctions between healthy and diseased states, ultimately allowing development of more potent therapeutics with fewer side effects.